Factors important to the secretion and matrix deposition of tissue transglutaminase


Data suggest that for TG2 to be secreted, an intact N-terminal FN binding site (for which TG2 has high affinity) is required, however interaction of TG2 with its high affinity binding partners presents both in the intracellular and extracellular space as well as with specific cell surface receptors may also be involved in this process. Using a site-directed mutagenesis approach, the effects of specific mutations of TG2 on its translocation to the cell surface and secretion into the ECM have been investigated. Mutations include those affecting FN binding (FN1), HSPGs binding (HS1, HS2) GTP/GDP binding site (GTP1, 2) as well as N-terminal and C-terminal domains (TG2 deletion mutants N, and C). By performing transglutaminase activity assays, cell surface protein biotinylation and verifying distribution of TG2 mutants in the ECM we demonstrated that one of the potential heparan sulfate binding site mutants (HS2 mutant) is secreted at the cell surface in a much reduced manner and is less deposited into the ECM than the HS1 mutant. The HS2 mutant showed a low affinity for binding to a heparin sepharose column demonstrating this mutation site may be a potential heparan binding site of TG2. Analogous peptides to this site were shown to have some efficiency in the inhibition of the binding of the FN-TG2 complex to cell surface heparan sulfates in a cell adhesion assay indicating the peptide to be representative of the novel heparin binding site within TG2. The GTP binding site mutants GTP1 and GTP2 exhibited low specific activity however, GTP2 showed more secretion to the cell surface in comparison to GTP1. The FN1 binding mutant did not greatly affect TG2 activity nor did it alter TG2 secretion at the cell surface and deposition into the ECM indicating that fibronectin binding at this site on the enzyme is not an important factor. Interestingly an intact N-terminus (?1-15) appeared to be essential for enzyme externalisation. Removal of the first 15 amino acids (N-terminal mutant) abolished TG2 secretion to the cell surface as well as deposition into the ECM. In addition it reduced the enzymes affinity for binding to heparin. In contrast, deletion of the C-terminal TG2 domain (?594-687) increased enzyme secretion to the cell surface. Consistent with the data presented in this thesis we speculate that TG2 must fulfill two requirements to be successfully secreted from cells. The findings indicate that the closed conformation of the enzyme as well as intact N-terminal tail and a novel HS binding site within the TG2 molecule are key elements for the enzyme’s localisation at the cell surface and its deposition into the extracellular matrix. The importance of understanding the interactions between TG2, heparan sulfates and other TG2 binding partners at the cell surface could have an impact on the design of novel strategies for enzyme inhibition which could be important in the control of extracellular TG2 related diseases.

Divisions: College of Health & Life Sciences > School of Biosciences
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Institution: Aston University
Last Modified: 28 Jun 2024 07:58
Date Deposited: 08 Feb 2012 13:20
Completed Date: 2011-03
Authors: Pytel, Kamila Malgorzata


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